The long range goal of this research is the elucidation of the allosteric mechanism of E. coli aspartate transcarbamylase. Work in the past has empahsized the thermodynamics of ligand binding and subuint interactions. We will extend our thermodynamic studies by determining free energies of interchain and intersubunit interactions by hydrogen exchange measurements and analytical gel chromatography, and by evaluating heat capacity changes accompanying ligand binding to the native enzyme and isolated subunits by batch calorimetry. The main thrust of future work, however, will be to correlate thermodynamics and structure, that is, to determine how the energy used for regulation is distributed throughout the structure. Three approaches will be taken. (a) The Gurd-Matthew static accessibility discrete charge model, or refinements of it, will be used together with the crystallographic coordinates to identify and characterize ionizable groups perturbed by ligand binding and subunit association. (b) Medium resolution hydrogen exchange methods will be developed and used to characterize changes in the molecular dynamics of ATCase and its subunits produced by effector binding and subunit association, and to identify regions of the structure perturbed by these interactions. (c) Single site mutants will be used to identify regions of the molecule involved in the transmission of allosteric signals. Selected mutants will also be characterized with respect to their electrostatics (by potentiometry and computer calculations), thermostability (by diffeerential scanning calorimetry), and molecular dynamics (by hydrogen exchange). The information obtained with these three approaches will be used to develop a quantitative model of the allosteric mechanism.